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PubMed1992

Effect of Plasmodium falciparum on blood feeding behavior of naturally infected Anopheles mosquitoes in western Kenya.

Feeding behavior was compared between infected and uninfected field-collected groups of Anopheles gambiae sensu lato and An. funestus from western Kenya. A significantly greater percentage (81%) of Plasmodium falciparum-infected An. gambiae s.l. females probed on experimental hosts (hamsters) than did uninfected females (38%). Among those females that initiated probing, there was no effect of infection status on the ability to take a bloodmeal. Plasmodium falciparum-infected An. gambiae s.l. probed more often (mean = 4.0) and for a longer time (mean = 277 sec) than did their uninfected counterparts (mean = 2.4 probes and mean probing time = 214 sec). Results for the small number of An. funestus that fed followed the same trend. Among infected An. gambiae s.l. females, there was no effect of sporozoite density on either the number of probes made or the total probing time. Among uninfected females, there was no difference in feeding behavior between nulliparous and parous females. In laboratory experiments, female age had no effect on blood-feeding behavior. Our findings provide evidence that natural malaria infection modifies the feeding behavior of Anopheles females.

Age Factors↗
PubMed1993

Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia.

This report describes an enzyme assay for the detection of Plasmodium falciparum. The assay is based on the observation that the lactate dehydrogenase (LDH) enzyme of P. falciparum has the ability to rapidly use 3-acetyl pyridine NAD (APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate. Human red blood cell LDH carries out this reaction at a very slow rate in the presence of APAD. We measured the development of APADH and found that the formation of this product could establish the basis of an assay that detected the presence of P. falciparum from in vitro cultures at parasitemia levels of 0.02%. We also had occasion to use this assay with clinical samples. We found a correlation between levels of parasitemia and the activity of parasite LDH. Parasite LDH (pLDH) activity could be measured in blood hemolysates and in plasma and serum from patients with malaria. We used the serum assay for pLDH and followed the level of pLDH in a patient with cerebral malaria prior to antimalarial treatment and during the recovery period. From these initial studies, it is evident that the measurement of pLDH has a correlation with parasitemia and may offer a method that can be developed into a simple test for the detection of Plasmodium parasitemia.

Animals↗
PubMed1994

Sporogonic development of cultured Plasmodium falciparum in six species of laboratory-reared Anopheles mosquitoes.

Sporogonic development of cultured Plasmodium falciparum was compared in six species of Anopheles mosquitoes. A reference species, A. gambiae, was selected as the standard for comparison. Estimates of absolute densities were determined for each lifestage. From these data, four aspects of parasite population dynamics were analyzed quantitatively: 1) successive losses in abundance as parasites developed from gametocyte to ookinete to oocyst stages, 2) oocyst production of sporozoites, 3) correlation between various lifestage parameters, and 4) parasite distribution. Parasite populations in A. gambiae incurred a 316-fold loss in abundance during the transition from macrogametocyte to ookinete stage, a 100-fold loss from ookinete to oocyst stage, yielding a total loss of approximately 31,600-fold (i.e., losses are multiplicative). Comparative susceptibilities in order were A. freeborni >> A. gambiae, A. arabiensis, A. dirus > A. stephensi, A. albimanus. The key transition(s) determining overall susceptibility differed among species. Despite species differences in oocyst densities and infection rates, salivary gland sporozoite production per oocyst (approximately 640) was the same among species. The most consistent association among lifestage parameters was a positive correlation between densities and infection rates of homologous lifestages. A curvilinear relationship between ookinete and oocyst densities in A. gambiae indicated a threshold density was required for ookinete conversion to oocysts (approximately 30 ookinetes per mosquito). The same relationship in A. freeborni was linear, with no distinct threshold. Ookinete and oocyst populations were negative binomially distributed in all species. Indices of heterogeneity in mosquito susceptibility to infection indicated that gene frequencies determining susceptibility fluctuated with time in all species, except A. freeborni where susceptibility remained homogenous throughout the study. This approach provides a framework for identifying mechanisms of susceptibility and evaluating Plasmodium sporogonic development in naturally occurring vector species in nature.

Analysis of Variance↗
PubMed1994

Characterization of naturally acquired human IgG responses against the N-terminal region of the merozoite surface protein 1 of Plasmodium vivax.

The primary structure of the merozoite surface protein 1 of Plasmodium vivax (PvMSP-1) revealed the existence of conserved and polymorphic blocks of the protein among different Plasmodium species. To characterize the naturally acquired IgG antibody responses to the PvMSP-1 molecule, the entire N-terminal portion of this protein was expressed as 10 overlapping glutathione S-transferase fusion proteins. The affinity-purified recombinant products were tested by enzyme-linked immunosorbent assay and Western blot against the sera of malaria patients from the state of Rondonia, Brazil. We found that the majority of these sera did not contain IgG antibodies recognizing recombinant proteins expressing exclusively interspecies conserved blocks of the molecule. In contrast, a high proportion of these same sera reacted against recombinant products expressing interspecies polymorphic regions of this protein. The poor B cell immunogenicity of the interspecies conserved blocks of the PvMSP-1 molecule most likely reflects important and unknown structural or functional features of these regions.

Adult↗
PubMed1995

Age-dependent carriage of multiple Plasmodium falciparum merozoite surface antigen-2 alleles in asymptomatic malaria infections.

Genetic diversity of the merozoite surface antigen-2 gene of the human malaria parasite Plasmodium falciparum has been analyzed in a Senegalese village where malaria is holoendemic. A cross-sectional survey of 65 residents was performed in 1992 during the high transmission season. Plasmodium falciparum was detected both by microscopy (77% positive samples) and DNA amplification using a single (29% or 38% positive samples, depending on the primers used) or nested polymerase chain reaction (PCR) (78% positive samples). The overlap between the positive nested PCR and microscopic examination was not complete. The PCR fragments were analyzed for size polymorphism on agarose gels, and were subsequently assigned to the major allelic families 3D7 or FC27 by hybridization with family-specific probes. Both allelic families were found, with a slightly higher prevalence for FC27. Chimeric alleles that failed to hybridize under stringent conditions to the reference probes were also observed. Some were typed using a novel PCR approach, using hybrid pairs of primers, consisting of a family-specific sense oligonucleotide combined with an antisense oligonucleotide specific for the other family. Combining typing techniques, 82% of the positive PCR results yielded more than one band. Both the overall number of fragments and the number of allelic types per carrier were markedly reduced around the age of 15 years. The number of DNA fragments decreased abruptly from an average of four per carrier before the age of 15 years to an average of two in individuals more than 15 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1996

Effect of pH on in vitro potency of amantadine against Plasmodium falciparum.

Amantadine is a monoprotic weak base that inhibits intraerythrocytic growth in in vitro cultures of Plasmodium falciparum, specifically chloroquine-resistant strains. Changes in the external PH of the medium are expected to result in a shift in the relative proportion of ionized and unionized species of amantadine by virtue of the weak base characteristic of the drug, influencing passage of the drug through the membrane. The ability of amantadine to alkalinize the food vacuole was determined using the accumulation of acridine orange as a vacuolar probe. Drug sensitivity following alteration of the pH gradient was assessed using the hypoxanthine method. Amantadine was able to alkalinize the food vacuole in the millimolar range; however, since its antimalarial activity is in the micromolar range, alkalinization of the food vacuole is not the primary action of the drug. The pH of the medium profoundly influenced susceptibility to chloroquine; the log of the 50% inhibitory concentration (IC50) values were linearly dependent on the external pH in both chloroquine-resistant and chloroquine-sensitive strains. Log IC50 values of amantadine exhibited a linear dependence on external pH in the chloroquine-sensitive strain, but in the chloroquine-resistant strain, a nonlinear parabolic function was found with the minimum IC50 at pH 7.03. Ammonium chloride did not interfere with the antimalarial activity of amantadine. The presence of the amine group on the hydrocarbon cage is essential for the activity of amantadine in Plasmodium falciparum. These results suggest factors in addition to pH gradient are involved in the effect of amantadine, possibly interactions with membrane phospholipids.

Adamantane↗
PubMed1996

Molecular cloning and antigenic mapping of heat-shock protein 70 from the malaria species Plasmodium bergheI.

We have isolated a 70-kD heat-shock protein (hsp-70) cDNA from Plasmodium berghei. A cDNA clone encoding the P. berghei hsp-70 was isolated and sequenced, demonstrating that it is highly homologous with other Plasmodium hsp-70s. One of the common features is a series of GGMP amino acid repeats at the carboxy terminus; there is also a long, AT-rich 5' untranslated region, a hallmark of other malarial RNAs. Hydropathy and antigenicity analyses suggest the presence of two hydrophilic domains. Recombinant peptides comprising different fragments of hsp-70 were expressed in Escherichia coli and assessed for antigenicity with antiserum from mice immunized with sonicated extracts of P. berghei. Antigenic sites map to regions that include the two hydrophilic domains.

Amino Acid Sequence↗
PubMed1997

Parasight F test compared with the polymerase chain reaction and microscopy for the diagnosis of Plasmodium falciparum malaria in travelers.

Imported malaria is an increasing problem worldwide. A rapid and accurate test for Plasmodium falciparum infection would facilitate the diagnosis of malaria in the returned traveler. The ParaSight F antigen capture assay (dipstick test) is a new diagnostic test for P. falciparum based on detection of circulating histidine-rich protein-2 antigen. We performed a blinded evaluation of this assay compared with microscopy and the polymerase chain reaction (PCR) for the detection of P. falciparum infection in 151 febrile travelers. Compared with the PCR, the dipstick test had a sensitivity of 88% and a specificity of 97%. The ability of the dipstick test to detect P. falciparum was similar with that of microscopy (88% versus 83%) since the species of Plasmodium in 14 of 133 malaria-infected patients could not be determined by microscopy due to low parasite numbers. The dipstick test was 40% sensitive for infections with < 50 parasites/microliter, 89% with 50-100 parasites/microliter, and > or = 93% with > 100 parasites/microliter. Circulating antigen was detectable in 68% of the patients seven days after initiation of treatment and in 27% at day 28. The dipstick test represents a simple and accurate test for the diagnosis of P. falciparum infection in the returned traveler.

Adolescent↗
PubMed1998

No influence of age on infection complexity and allelic distribution in Plasmodium falciparum infections in Ndiop, a Senegalese village with seasonal, mesoendemic malaria.

We have shown previously that in Dielmo, a Senegalese village with intense perennial Plasmodium falciparum transmission, the infection complexity and the distribution of some allelic types harbored by asymptomatic carriers was age-dependent. We report here an investigation of these parameters in Ndiop, a village located 5 km from Dielmo, where malaria is mesoendemic and seasonal, and where immunity is acquired at a very low rate, as indicated by the lifelong distribution of P. falciparum clinical attacks. Blood was collected from 143 and 125 inhabitants, including 122 individuals sampled in both surveys, during two cross-sectional surveys at one-month intervals during the 1994 transmission season. Plasmodium falciparum parasites were genotyped for three polymorphic single copy genes. Genetic diversity was very large, with 17, 43, and nine distinct alleles detected for the merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein loci, respectively. These figures, similar to those previously observed in Dielmo, indicate that the parasite genetic diversity is not directly related to the inoculation rate, at least in the range of transmission intensity studied here. The complexity of the asymptomatic infections (average number of distinct genotypes per isolate) was more than two-fold lower in Ndiop than in Dielmo and importantly, did not decrease with age. Likewise, the allele distribution was not influenced by age, contrasting with the observations made in Dielmo. This indicates that the number of parasite types per isolate and the influence of age on complexity and allele distribution depend on the level of endemicity, consistent with the interpretation that they reflect acquired anti-parasite immunity.

Adolescent↗
PubMed1999

Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand.

Parasite genotyping by the polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in a Karen population resident on the northwestern border of Thailand where malaria transmission is low (one infection/person/year). Plasmodium falciparum infections were genotyped for allelic variation in three polymorphic antigen loci, merozoite surface proteins-1 and -2 (MSP-1 and -2) and glutamaterich protein (GLURP), before and after antimalarial drug treatment. Population genotype frequencies were measured to provide the baseline information to calculate the probability of a new infection with a different or the same genotype to the initial pretreatment isolate. Overall, 38% of the infections detected following treatment had an identical genotype before and up to 121 days after treatment. These post-treatment genotypes were considered recrudescent because of the low (< 5%) probability of repeated occurrence by chance in the same patient. This approach allows studies of antimalarial drug treatment to be conducted in areas of low transmission since recrudescences can be distinguished confidently from newly acquired infections.

Alleles↗
PubMed2000

Assessing drug sensitivity of Plasmodium vivax to halofantrine or choroquine in southern, central Vietnam using an extended 28-day in vivo test and polymerase chain reaction genotyping.

Chloroquine-resistant Plasmodium vivax malaria is emerging in Oceania, Asia, and Latin America. We assessed the drug sensitivity of P. vivax to chloroquine or halofantrine in two villages in southern, central Vietnam. This area has chloroquine-resistant Plasmodium falciparum but no documented chloroquine-resistant P. vivax. Standard dose chloroquine (25 mg/kg, over 48 hours) or halofantrine (8 mg/kg, 3 doses) was administered to 29 and 25 patients, respectively. End points were parasite sensitivity or resistance determined at 28 days. Of the evaluable patients, 23/23 100% (95% confidence interval [CI] 85.1-100) chloroquine and 21/24 (87.5%) (95% CI 67.6-97.3) halofantrine-treated patients were sensitive. Three halofantrine recipients had initial clearance but subsequent recurrence of their parasitemias. Genotyping of the recurrent and Day 0 parasitemias differed, suggesting either new infections or relapses of liver hypnozoites from antecedent infections. Among these Vietnamese patients, P. vivax was sensitive to chloroquine and halofantrine. Genotyping was useful for differentiating the recurrent vivax parasitemias.

Adolescent↗
PubMedDate not supplied

Short report: failure to select for chloroquine- or mefloquine-resistant Plasmodium berghei through drug pressure in Anopheles stephensi mosquitoes.

We investigated whether chloroquine- or mefloquine-resistant Plasmodium berghei could be selected through drug pressure applied during continuous cyclical transmission in Anopheles stephensi mosquitoes. Mosquitoes were infected by feeding them on mice previously inoculated with a drug-sensitive clone of P. berghei ANKA. Mosquitoes ingested mefloquine or chloroquine with the infectious blood-meal, or by feeding on a drug-treated (uninfected) mouse 4 or 10 days after the infectious blood-meal. Twenty-two days after being infected, mosquitoes transmitted sporozoites to uninfected mice. Blood from these animals was used to infect naive mice that were then used to reinitiate the mouse/mosquito/mouse cycle. A total of 20 passages through mosquitoes were completed while under drug pressure. Drug-resistance levels were assessed in the initial clone and after 20 passages through mosquitoes. None of 18 "sub-clones" of parasites showed significant increases in chloroquine or mefloquine resistance, suggesting that exposure of sporogonic stage Plasmodium to chloroquine or mefloquine will not result in the development of drug resistance.

Animals↗
PubMedDate not supplied

Placental changes associated with fetal outcome in the Plasmodium coatneyi/rhesus monkey model of malaria in pregnancy.

Term placentas collected surgically from seven Plasmodium coatneyi-infected rhesus monkeys, one abortion, and five controls were evaluated histopathologically. The placentas from Plasmodium-infected dams had more significant pathologic changes than those from controls for six parameters (P < 0.05) and higher numbers of activated (LN5 + Zymed) macrophages in the intervillous space (IVS) (P = 0.0173). Total parasite load (TPL) was defined as the sum of all weekly peripheral infected red blood cell counts for each trimester and for the entire pregnancy. High first trimester PLs were more likely to result in fetal demise (P = 0.0476) or increased placental damage in surviving infants. As trimester 2-3 TPL increased, so did the number of activated macrophages (P < 0.05) and the total malaria pigment scores (P < 0.05). Low birth weight (LBW) and intrauterine growth retardation (IUGR) were associated with high pigment scores and high numbers of activated macrophages in the IVS. High placental damage scores were not associated with IUGR, LBW, or early infant mortality.

Animals↗
PubMedDate not supplied

A trial for a DNA diagnosis of Plasmodium vivax malaria recently reemerging in the Republic of Korea using microtiter plate hybridization assay.

The polymerase chain reaction-based microtiter plate hybridization (PCR-MPH) assay was utilized for a DNA diagnosis of Plasmodium vivax malaria, which has recently reemerged in the Republic of Korea. The subjects were 18 parasite-proven patients and 5 healthy controls. Follow-up blood samples were collected from 4 patients after a standard course of treatment. Polymerase chain reaction and electrophoresis of all the patients' blood showed a prominent band at the 138 base pair area, but not in the controls or after treating the patients. Hybridization of the PCR products with known species-specific probes of the 18S rRNA of various malaria species revealed strong positive reactions against the Plasmodium vivax-specific probe (absorbance 1.30-1.90 at 405 nm) in all of the patients. The absorbance was positively correlated with the degree of blood parasitemia, but with a borderline significance. Sequencing of the probe region of the Korean P. vivax revealed no significant variations from the typical P. vivax. The results show that the PCR-MPH is a highly useful technique for the DNA diagnosis of Korean vivax malaria.

Adult↗
PubMed2002

A retrospective examination of sporozoite-induced and trophozoite-induced infections with Plasmodium ovale: development of parasitologic and clinical immunity during primary infection.

A retrospective analysis was made of clinical and parasitologic parameters in patients with induced Plasmodium ovale infection to document the initial clinical and parasitologic response and their subsequent development of clinical and parasitologic immunity, and to determine the effect of previous homologous and heterologous malaria on subsequent infection with this parasite. The prepatent periods were relatively uniform. Eight patients injected with sporozoites that had been stored frozen had a median prepatent period of 14 days (range = 14-20 days). Thirty-five patients infected via the bites of infected mosquitoes had a median prepatent period of 15 days (range = 12-18 days). In eight patients previously infected with P. vivax, the median prepatent period was 16 days. High-intensity fever (> or = 104 degrees F) was frequently seen, with instances of fever > or = 106 degrees F recorded on many occasions. Fever > 101 degrees F and > 104 degrees F occurred for much shorter periods of time than had been observed in patients infected with P. falciparum. Parasite counts > 10,000/microL were infrequent; in most patients, such parasite counts rarely lasted more than two or three days. Gametocytemia was generally of low density and lasted only a few days. The overall length of the clinical and parasitologic period was much shorter compared with that seen in patients infected with P. falciparum. Previous infection with P. ovale did not prevent reinfection, but resulted in reduced levels of parasitemia and fever. Previous infection with heterologous species of Plasmodium did not prevent infection; some reduction in the frequency and intensity of fever and parasite counts was evident. Previous infection with homologous or heterologous parasites failed to eliminate the production of infective gametocytes. A total of 462 lots of mosquitoes were fed on 67 patients with no previous history of infection. Of these feedings, 168 (36.4%) resulted in infection as determined by the presence of oocysts on the midguts of dissected mosquitoes. As shown, the infection rate increased with the density of gametocytes even though 48 (23.4%) of 205 lots of mosquitoes fed when no gametocytes were detected were infected.

Animals↗
PubMed2005

Aotus lemurinus griseimembra monkeys: a suitable model for Plasmodium vivax sporozoite infection.

This study describes a successful Plasmodium vivax sporozoite infection in Aotus lemurinus griseimembra. Twenty-eight naive or previously infected monkeys, either splenectomized or spleen intact, were inoculated intravenously or subcutaneously with Plasmodium vivax sporozoites of the Salvador I strain or with two wild isolates (VCC-4 and VCC-5; Vivax-Cali-Colombia). The monkeys were successfully infected regardless of the parasite strain, spleen presence, or inoculation route and showed prepatent periods that ranged from 16 to 89 days. Only one monkey inoculated intravenously failed to develop parasitemia. Since immune protection against malaria pre-erythrocytic forms is mediated by both helper and cytolytic T cells that may home in the spleen and P. vivax cultures are not yet available; the use of spleen-intact A. lemurinus griseimembra, susceptible to both adapted and non-adapted strains of P. vivax sporozoites, is a valuable model for evaluation of pre-erythrocytic vaccine candidates.

Animals↗
PubMed2005

Antigenicity, immunogenicity, and protective efficacy of Plasmodium vivax MSP1 PV200l: a potential malaria vaccine subunit.

The merozoite surface protein 1 (MSP-1) is expressed in all Plasmodium species and is considered a major malaria vaccine candidate. We found that MSP-1 from Plasmodium vivax (PvMSP-1) contains a region of significant sequence homology with the 190L subunit vaccine derived from the P. falciparum MSP-1. The fragment, termed Pv200L, was expressed as a recombinant protein in Escherichia coli (rPv200L) and used to asses its immunologic relevance as a vaccine target. A cross-sectional, seroepidemiologic study conducted in Buenaventura, Colombia showed that 52.2% (95% confidence interval [CI] = 39.8-64.3) of individuals previously exposed to P. vivax and 72.8% (95% CI = 61.8-82.1) of P. vivax-infected patients had IgG antibodies to rPv200L. Immunization of BALB/c mice and Aotus monkeys induced IgG antibodies (titer > 10(6)) that cross-reacted with P. vivax parasites. Immunized monkeys displayed partial protection against a challenge with P. vivax blood stages. Our results suggest that Pv200L is a new malaria vaccine subunit and deserves further testing.

Amino Acid Sequence↗
PubMed2003

Infectivity of Plasmodium falciparum gametocytes in patients attending rural health centres in western Kenya.

BACKGROUND: Experimentally studying the transmission of the malaria parasite and its regulating factors requires availability of human blood donors carrying infectious gametocytes. The difficulty of identifying gametocyte carriers from the community is often limited due to financial and human resources constraints. The available alternative is rural health centres where malaria patients go for treatment. In this study, the potential of recruiting volunteers and acquiring infectious blood for experimental infections from rural health centers in malaria endemic area was examined through routine patient diagnosis. OBJECTIVE: To examine the patients presenting at rural health centers for the potential to carry sexual stage malaria parasite and test their infectivity to Anopheles gambiae mosquitoes. SETTING: Mbita Health Centre, Mbita Town Ship, Suba District, western Kenya. METHODOLOGY: Routine survey of all patients attending Mbita Health Centre with suspected malaria. Patients were examined for Plasmodium falciparum trophozoites and gametocytes. Gametocyte-positive volunteers were recruited for their potential to infect Anopheles mosquitoes via membrane feeding. RESULTS: Three thousand nine hundred and eighty seven patients were screened between May 2000 and April 2001. Plasmodium falciparum was the predominant parasite species and P. malariae being the only minor species, accounting for 0.9% of malaria cases. Clinical malaria varied with age and prevailed throughout the year with a slight seasonality. Gametocyte prevalence was low (0.9-6.6%), and gametocyte densities were generally very low with a geometric mean of 39 gametocytes per microl blood. Children aged > 5 years constituted 67% of all gametocyte carriers. Only 22 volunteers with mean gametocytes density of 39.62 per microl blood (range: 16-112) were recruited for study of parasite infectiousness to laboratory-reared mosquitoes. Only two patients infected 1% of 1099 mosquitoes with one or two oocysts. CONCLUSION: The low gametocyte densities or other possible host and vector related factors regulating infectivity of gametocyte carriers to mosquitoes may have caused the poor infections of mosquitoes. This study indicates that rural health centers in malaria-endemic areas may not be suitable for recruiting infectious gametocyte donors for studies of vector competence. They are suitable for passive clinical case surveillance and for evaluation of the effects of control measures.

Adolescent↗